Formation of glutathione conjugates of propranolol in situ by guinea pig microsomes supplemented with GSH-S-transferase and GSH was based on two criteria. 1) After treatment with Alpha-glutamyltranspeptidase, the glutathione conjugate detected in a HPLC system was converted to another substance having a shorter retention time than that treated with BSA. 2) Double label experiment using 14C-GSH and 3H propranolol (4-3H) demonstrated unequivocally that the conjugate peak detected in the HPLC system is glutathione conjugate. Since we have previously found that the decrease in the formation of 50HPr correlated with the formation of a GSH-conjugate in liver microsomes from BNF treated rats, it seems likely that the GSH-conjugate and 50HPr are derived from a common intermediate, perhaps propranolol-5,6-oxide. It is noteworthy that the presence of GSH plus GSH transferase caused the lowering of 5-hydroxypropranolol formation in all microsomes from stomach, small intestine and colon, suggesting an important role of GSH-transferase in GI tract for the inactivation of the epoxide.